Gorse (Ulex europeaus) wastes with 5,6-dimethyl benzimidazole supplementation can support growth of vitamin B12 producing commensal gut microbes

Many commensal gut microbes are recognized for their potential to synthesize vitamin B12, offering a promising avenue to address deficiencies through probiotic supplementation. While bioinformatics tools aid in predicting B12 biosynthetic potential, empirical validation remains crucial to confirm production, identify cobalamin vitamers, and establish biosynthetic yields. This study investigates vitamin B12 production in three human colonic bacterial species: Anaerobutyricum hallii DSM 3353, Roseburia faecis DSM 16840, and Anaerostipes caccae DSM 14662, along with Propionibacterium freudenreichii DSM 4902 as a positive control. These strains were selected for their potential use as probiotics, based on speculated B12 production from prior bioinformatic analyses. Cultures were grown in M2GSC, chemically defined media (CDM), and Gorse extract medium (GEM). The composition of GEM was similar to CDM, except that the carbon and nitrogen sources were replaced with the protein-depleted liquid waste obtained after subjecting Gorse to a leaf protein extraction process. B12 yields were quantified using liquid chromatography with tandem mass spectrometry. The results suggested that the three butyrate-producing strains could indeed produce B12, although the yields were notably low and were detected only in the cell lysates. Furthermore, B12 production was higher in GEM compared to M2GSC medium. The positive control, P. freudenreichii DSM 4902 produced B12 at concentrations ranging from 7 ng mL−1 to 12 ng mL−1. Univariate-scaled Principal Component Analysis (PCA) of data from previous publications investigating B12 production in P. freudenreichii revealed that B12 yields diminished when the carbon source concentration was ≤30 g L−1. In conclusion, the protein-depleted wastes from the leaf protein extraction process from Gorse can be valorised as a viable substrate for culturing B12-producing colonic gut microbes. Furthermore, this is the first report attesting to the ability of A. hallii, R. faecis, and A. caccae to produce B12. However, these microbes seem unsuitable for industrial applications owing to low B12 yields.


Introduction
The biological role and molecular structure of vitamin B 12 Vitamin B 12 is involved in various enzymatic reactions, including isomerases, methyltransferases, and dehalogenases [1], with B 12 -dependent dehalogenases being exclusive to bacteria [2].It is a critical cofactor in the type 1 reaction, catalysing the isomerization of L-methylmalonyl-CoA to succinyl-CoA, that subsequently enters the Krebs cycle [3].Furthermore, B 12 is crucial for the type 2 reaction of methyl transfer from 5-methyltetrahydrofolate to homocysteine, to produce tetrahydrofolate and methionine [4].Hindrance in this type 2 reaction owing to B 12 deficiency results in the malformation of red blood corpuscles, and manifests as pernicious anaemia [5].
The bioactivity of vitamin B 12 hinges on three critical ligands, namely, the central cobalt ion located in the upper corrin ring, the methyl side-group, and 5,6-dimethylbenzimidazole (DMB) (see Fig 1).The methyl side-group and DMB are attached to the cobalt ion with a coordinate bond.The cobalt ion switches from the oxidised intermediate (III) to the reduced (I) state during the transfer of the methyl-group to homocysteine to form methionine.The oxidised cobalamin is subsequently re-alkylated with n-methyl-THF.The exact mechanism has been described previously by Matthews et al. [6].
Dietary vitamin B 12 however is generally present with the methyl, adenosyl, hydroxyl or cyano group associated with the central cobalt.The cyano vitamer is a semi-synthetic stable derivative of B 12 commonly used in dietary and medical applications [7].Other variants such as azidocobalamin, sulfocobalamin or nitrocobalamin are derivatives synthesised as intermediates for analytical purposes [8].Regardless of the ingested vitamer, once B 12 is absorbed in the body, it is converted to the hydroxyl-form, that is then re-alkylated to the methyl form; ultimately entering the methionine synthesis pathway as described previously [4].There is variation in the second ligand moiety, where either the biologically active DMB, or its structural homologue, adenyl-ribofuranose phosphate, may be present.Only the DMB-based vitamer is capable of participating in the catalytic reaction facilitated by methionine synthase.

Harnessing microbes for industrial B 12 production
The biosynthesis of B 12 requires several enzymes, that are only found in the bacteria and archaea domains of life [9,10].The complete set of genes required for de novo production of cobalamins; particularly B 12 , has been observed in numerous bacteria such as Pseudomonas denitrificans, Bacillus megaterium, Rhodobacter capusulatus, and Thermosipho melanesiensis, to name a few [11].Many other bacteria such as Escherichia coli, Kosmotoga olearia and Fervidobacterium nodosum can only use salvage pathways to synthesise cobalamin from precursors.The population of these microbes capable of cobalamin production through salvage pathways plays an important role in the micro-ecology of the niche they inhabit, firstly, through commensalistic or even symbiotic association with primary B 12 producers [9], and secondly, by facilitating tertiary associations with other microbes heterotrophic for the precursor.In industrial setups however, candidate selection of B 12 producers is largely influenced by the genetic stability of the strain, bioconversion efficiency, B 12 yields, extracellular production capability, and rapid turnover.
Many microbial species; either naturally, or through genetic engineering, are capable of over-synthesising vitamin B 12 .The most commonly used species in industry are Propionibacterium freudenreichii, Propionibacterium shermanii and Pseudomonas denitrificans.Growth conditions are generally aerobic as de novo biosynthesis of vitamin B 12 is an energetically demanding process, particularly the ligand DMB [9].Consequently, there is great variability in the substrates used depending on the metabolic capability of the microbe employed.For example, many media formulations use additional supplementation of cobalt, DMB, and in rare cases, haeme [12].This helps cope with the increased demand for micronutrients and alleviates the metabolic burden of de novo DMB or corrin synthesis.Interestingly, this has lead to investigations on waste stream valorisation with cobalt and DMB supplementation for the growth of B 12 producing bacteria.For example, P. freudenreichii has been shown to grow on apple pomace and potato wastes for large scale production of propionic acid, with vitamin B 12 obtained as a secondary product in significant quantities [13,14].Such endeavours help generate high-value products and circularise waste streams otherwise fated for landfill or wastewater treatment plants.

B 12 from Gorse wastes using gut microbes
Gorse (Ulex europaeus) is a shrub belonging to the Fabaceae family that has become invasive in many parts of the world [15].However it is rich in proteins and plant-bioactives, and a leaf protein extraction process was previously described to effectively utilise the biomass [16].The process generated a protein depleted fraction (PDF) that contained residual proteins, plant bioactives, and carbohydrates as a by-product.In an effort to valorise this waste stream, three butyrate-producing microbial candidates, namely Anaerobutyricum hallii DSM 3353, Roseburia faecis DSM 16840, and Anaerostipes caccae DSM 14662, were investigated for their ability to grow on media comprising of PDF as the sole carbon and nitrogen source.These microbial candidates were chosen due to their potential to produce vitamin B 12 [17] and their purported beneficial impact in the human gut [18,19].Propionibacterium freudenreichii DSM 4902, was used as a positive control for vitamin B 12 production.

Materials and methods
All chemicals were purchased from Merck, (Darmstadt, Germany) unless stated otherwise.All water used was from the in house Milli-Q 1 purification system.All experiments were performed in triplicate.

Media
Media preparation was carried out in an anaerobic cabinet (Don Whitley Scientific, West Yorkshire, UK) set for 10% H 2 , 10% CO 2 , and 80% N 2 , with Milli-Q water which was first flushed with N 2 and autoclaved.The water was then allowed to equilibrate in the cabinet prior to preparation for 48 h.

Media preparation
M2GSC.The recipe for M2GSC is provided in section 1.1 of S1 File, and was prepared as described previously by Cummings and Macfarlane [20].The medium was allowed to equilibrate in the anaerobic cabinet for 48 h before use.
Chemically defined medium (CDM).CDM was prepared as described previously by Soto-Martı ´n et al [17], except with the inclusion of 5,6-dimethylbenzimidazole (DMB, 20 mg L −1 ).The medium was filter-sterilised using 0.2 μm filter (Millipore, Merck, US) and allowed to equilibrate in the anaerobic cabinet.The composition of the CDM is provided in section 1.2 of S1 File.
Gorse extract medium (GEM).GEM was prepared by replacing the carbon and nitrogen source of CDM with the Gorse protein-depleted fraction (PDF).The composition of PDF is provided in a previous publication [16].It contained residual protein (53.9 mg g −1 dry mass) and polysaccharides / sugars (glucose equivalent of 88.9 mg g −1 dry mass).Given that the plant mass was previously treated with cellulase as part of the protein extraction process, the carbohydrate profile of the PDF was expected to comprise of low molecular weight polysaccharides and glucose.Other monomers such as xylose, fucose, and glucuronic acid were expected to remain largely absent as xylanase, pectinase and other cell-wall digesting enzymes were not employed in the process.
The PDF was used at a final concentration of 25 g L −1 .This meant that GEM contained about 1.35 g L −1 of proteins and 2.3 g L −1 of carbohydrates; values comparable to CDM.
Culture revival and growth.Microbial species were revived from frozen stocks using M2GSC (5 mL) in Hungate tubes (sealable with rubber caps) in anaerobic conditions and incubated at 37˚C for five days.The strains were sub-cultured by inoculating 0.5 mL of the culture in 4 mL of fresh M2GSC and incubated for two days.Growth was measured with optical density at 650 nm using Novaspec-II visible spectrophotometer (Pharmacia LKB Biotechnology AB, Uppsala, Sweden).
Production of B 12 samples.From the revived stock, 200 μL was inoculated in triplicate in M2GSC, CDM or GEM (10 mL) and gently mixed through repeated aspiration.The culture was statically incubated at 37˚C for 48 h.Sample (1 mL) was drawn at time points 0 h, 6 h, 12 h, 24 h, 30 h, 36 h and 48 h and centrifuged at 10 000 × g for 20 min at 4˚C (Biofuge (Fresco), Heraeus Instruments, Germany).The supernatant was recovered and analysed for B 12 content.The pellet was resuspended in PBS buffer (1 mL) and slowly cooled to −80˚C by first placing them in an ice-bath during sample collection, followed by incubation in −20˚C freezer overnight.Samples were then stored in a −80˚C freezer for a couple of days.This was performed to facilitate gradual growth of ice crystals to pierce and rupture the bacterial cell wall.Samples were then thawed to 4˚C overnight and sonicated (MSE Soniprep 150, UK) in two 10 s bursts to rupture the cells.The cells were centrifuged at 10 000 × g for 20 min and the supernatant was recovered and analysed for B 12 content.

Literature review
To retrieve previous literature on B 12 production in P. freudenreichii, the search structure: (propionibacterium AND (cobalamin OR B12)) was used in PubMed and Scopus.The relevant titles were selected and analysed by eye to obtain values for B 12 yield, the carbohydrate concentration used (Carbon gL −1 ), fermentation time (Time h), cobalt salt used (mg L −1 ) and added DMB (mg L −1 ).These values were used for the construction of a univariate-scaled PCA model to help compare and contextualise the B 12 yields obtained in the work described herein.The details of the study and B 12 are provided in S1 Table in S1 File.

Statistical analysis
All statistical analyses were performed using R (4.2.2) [22] using the tidyverse (2.0.0) [23] package for data processing and visualisation.Normality of the data distribution was checked using the Shapiro-Wilks test.The Carbon (g L −1 ), Time (h), and B 12 (mg L −1 ) values were log 10 transformed to achieve normality.
The k-means analysis followed by univariate-scaled principal component analysis was performed using the cluster (2.1.4)[24], and factoextra (1.0.7) [25] packages.A dimension reduction model was made using previously published B 12 yields from P. freudenreichii.A k-means analysis (MacQueen algorithm) was performed to observe clustering in the raw data.The raw data was then subjected to a univariate-scaled PCA analysis and the clusters established from k-means analysis was overlaid on the plot to observe their relative positions.This PCA model was then used to interpolate the relative position of the data points representing the experimental conditions described in our study.This was used to understand which cluster of experimental conditions our setup matched and compare the corresponding B 12 yields.The raw data and codes can be found at https://doi.org/10.17605/osf.io/3yb2r to reproduce the analyses.In previous experiments by Soto Martı ´n et al. [17], A. caccae DSM 14662 and R. faecis DSM 16840 were grown in CDM variants to assess trophic capabilities.They were initially grown in a semi-defined, vitamin-controlled medium using vitamin-free casein acid hydrolysate fortified with L-tryptophan, L-serine, L-threonine, L-glutamine, and L-asparagine to check for auxotrophy for each individual vitamin.Subsequently, they were grown in an amino-acid controlled CDM supplemented with all vitamins to check for auxotrophy for each amino acid.All three strains used here were able to grow well in both versions of CDM used by by Soto-Martı ´n et al [17].The growth profile in the panel for CDM in Fig 2 differs from previous reports, and may be explained by the pre-culture conditions.Soto-Martı ´n et al subjected the microbes through two passages in CDM before measuring their growth, while in this study, the microbes were revived in M2GSC and directly inoculated in CDM.

Growth profiles
The GEM and CDM used were identical in their base composition of added vitamins (as well as absence of natural cobalamins), minerals, micronutrients, and precursors.The key difference lay in their carbohydrate and protein sources.While GEM contained residual proteins and peptides, CDM contained purified amino acids.This difference may have contributed to the lack of growth by A. caccae DSM 14662 and R. faecis DSM 16840 in CDM.Certain bacterial species are known to prefer peptides as a nitrogen source rather than individual amino acids [26] under anaerobic conditions [27].The uptake of individual amino acids is energetically demanding and requires active Na 2+ mediated transmembrane transport [28] and is the primary rate-limiting step in nitrogen uptake [27].A. caccae DSM 14662 and R. faecis DSM 16840 are typically cultured in YCFA medium [29,30] and the cobalamin-devoid, free-amino-acid based CDM may have been energetically incompatible for their growth.

B 12 production in experimental species
Vitamin B 12 yields of the experimental and control bacterial candidates is shown in Table 1 below.
Vitamin B 12 was below detection levels at the 0 h time point across all tested media.P. freudenreichii DSM 4902 and R. faecis DSM 16840 were able to produce B 12 at the 48 h end-point in M2GSC as shown in Table 1.P. freudenreichii DSM 4902 showed B 12 in the supernatant as well as the cell lysate fraction.
Vitamin B 12 was detected in the lysates of A. caccae DSM 14662 and A. hallii DSM 3353 cultured in the GEM medium.In contrast, no B 12 was observed in M2GSC, despite it being the ideal growth medium.DMB was exclusively added to GEM and was likely critical to B 12 production.The growth of A. caccae and A. hallii in M2GSC without detectable B 12 in the lysates could be attributed to the synthesis of pseudo-B 12 vitamers, that were not detectable by the LC/ MS method employed in the study.This may also explain the lowered B 12 levels in M2GSC compared to GEM for P. freudenreichii DSM 4902 and R. faecis DSM 16840.Although these microbes demonstrated the ability to synthesize biologically active vitamers in anaerobic conditions, the addition of DMB likely alleviated the metabolic burden required for de novo synthesis, resulting in an increased total B 12 production in GEM compared to M2GSC.In this experiment, B 12 measurement was performed using LC/MS, capable of detection at ng quantities in the sample.Physiologically, such quantities are much higher than the metabolic requirement of the cells [31].This suggests that the chosen bacterial species were able to overproduce B 12 , but the experimental conditions, or perhaps the media design was unsuitable for any real commercial application.Moreover, this helps confirm [32] that A. hallii (formerly E. hallii) could produce B 12 vitamers in the gut.However, the three experimental microbes are unviable sources of B 12 for industrial applications.
Next, for a strain to be viable candidate for probiotic applications towards B 12 supplementation, it requires to secrete B 12 in sufficiently large quantities in the extracellular environment.In all the three experimental gut microbes however, B 12 was only detected in the cell lysates.This makes them unsuitable for application as probiotic B 12 supplements.
Lastly, of particular concern was the observed low B 12 yields for P. freudenreichii DSM 4902, which served as the positive control for this experiment.Generally, P. freudenreichii DSM 4902 has yields �0.2 μg mL −1 , yet the corresponding values noted in Table 1 were about 13× lower.A comparative analysis with other literature investigating P. freudenreichii for B 12 production was conducted to explore potential causes.

Component analyses using previous literature
The systematic online searches yielded 1651 hits from which 230 research titles were retrieved post title screening.A final list of 36 titles were selected based on the relevance of the research described therein.The data points for the concentration of the carbon source (gL −1 ), cobalt (mg L −1 ), DMB (mg L −1 ) and total fermentation time (h) were retrieved and is presented in S1 Table in S1 File.This only included cases of batch culturing of P. freudenreichii.
The data in S1 Table in S1 File was first subjected to k-means clustering to look for grouping patterns in the various experimental conditions that may correspond to B 12 yields.Based on the "elbow method", three groups were deemed ideal to explain clustering in the data (S2 File).Next, the data was subjected to a univariate-scaled PCA analysis and the corresponding grouping schema was overlaid to visualise their clustering on the PCA plot, as shown in Fig 3.
In Fig 3, the PCA model could account for 80.6% of the total observed variance in the data across Dimensions 1 and 2. The factors in the model, namely Carbon, Cobalt, DMB and Time accounted for 15.7%, 37.9%, 36.8% and 9.6% of the variance accounted in Dimension 1.In Dimension 2, Carbon, Cobalt, DMB and Time accounted for 39.4%, >0.1%, 0.1% and 60.6% of the variance.
Cluster 1 represents conditions with media employing high concentrations of carbon source, as well as a longer fermentation time, Cluster 2 represents the absence of precursors and low carbon source concentrations, and Cluster 3 represents high use of the precursors.
The point labelled "E" represents the experimental conditions and plots the closest to the mid-point of Cluster 2 (d = 0.718) compared to Clusters 1 (d = 2.57) and 3 (d = 5.86), where d represents the Euclidean distance between the points.This implies that the experimental condition described in this work was similar to that represented by Cluster 2. The median value of the conditions represented in each cluster is presented in Table 2.
The yield in Cluster 2 (0.2 mg L −1 ) was significantly lower than in Clusters 1 (5.7 mg L −1 ) and 3 (6.7 mg L −1 ), respectively (F (2,44) = 5.298, p = 0.009).These findings suggest that the availability of carbon source (via carbohydrates), was lower in the experimental setup described here compared to previous publications.As presented in Table 1, B 12 content was predominantly retained within the cell lysate, indicating that the microbes did not secrete B 12 extracellularly.Consequently, to achieve desired B 12 yields, it is imperative to supply essential nutrients and precursors at sufficiently high concentrations, as indicated by the values presented in Table 2. S1 Fig illustrates boxplots comparing the levels of Cobalt, Carbon, DMB, and Time for each cluster.
Thus the data from Table 2 suggest that the yields of B 12 are impacted when the carbohydrate concentration in the media is > 30 g L −1 .Previously reported experimental conditions were clustered using k-means analysis.The clustering scheme was overlaid on a univariate-scale PCA model that was constructed using previously reported parameters for the growth of Propionibacterium freudenreichii, namely, 5,6-dimethylbenzimidazole (DMB, mg L −1 ), cobalt (mg L −1 ), total carbohydrates (Carbon, g L −1 ), and incubation time (h).The point marked 'E' refers to the intrapolated position of the experimental conditions described herein and correspondingly, the expected B 12 yields, which appears to associate to cluster 2. Ellipses represent 95% confidence limits of each cluster.Data and codes used for dimension reduction analyses is provided in S2 File. https://doi.org/10.1371/journal.pone.0290052.g003

Fig 1 .
Fig 1. Structure of cobalamin vitamers.Variable side-groups (S v ) form coordinate bonds with the central cobalt ion.The ligands at location L l determine if the cobalamin vitamer is biologically active in humans with 5,6-dimethybenzimidazole (DMB) or inactive with adenine (marked in red).The cyano-group (marked in blue) is an example of semi-synthetic cobalamin derivates.B 12 vitamers can be found with combinations of S v and L l ligands.https://doi.org/10.1371/journal.pone.0290052.g001

Four
bacterial strains, namely, A. caccae DSM 14662, A. hallii DSM 3353, R. faecis DSM 16840 and P. freudenreichii DSM 4902 were incubated in CDM, GEM and M2GSC over a period of 48 h.The growth profiles are shown in Fig 2. All tested strains demonstrated robust growth in M2GSC and GEM media, reaching ΔO.D values >1.0.In contrast, CDM medium only supported the growth of A. hallii DSM 3353 and P. freudenreichii DSM 4902, with ΔO.D values <0.51.

Fig 3 .
Fig 3. Dimension reduction of experimental conditions to elucidate their influence on B 12 yields.Previously reported experimental conditions were clustered using k-means analysis.The clustering scheme was overlaid on a univariate-scale PCA model that was constructed using previously reported parameters for the growth of Propionibacterium freudenreichii, namely, 5,6-dimethylbenzimidazole (DMB, mg L −1 ), cobalt (mg L −1 ), total carbohydrates (Carbon, g L −1 ), and incubation time (h).The point marked 'E' refers to the intrapolated position of the experimental conditions described herein and correspondingly, the expected B 12 yields, which appears to associate to cluster 2. Ellipses represent 95% confidence limits of each cluster.Data and codes used for dimension reduction analyses is provided in S2 File.